Determination of bromhexine in plasma by reversed-phase liquid chromatography. Interference of lipoproteins on extraction.

Extraction of the hydrophobic tertiary amine bromhexine from plasma using cyclohexane-heptafluorobutanol (99.5:0.5, v/v) was studied at different pH values. The extraction yield from buffer solutions was quantitative at pH greater than 4.1, but from plasma the extraction yield decreased with increasing pH. Furthermore, at pH 8.4 the extraction yield varied greatly (56-99%) in different human plasma. The addition of lipoproteins to phosphate buffer, at pH 8.1, decreased the extraction yields considerably. Quantitative extraction from plasma was obtained by using a very long extraction time at pH 8.4 or by decreasing the pH to 5.4. The chromatographic system consisted of a reversed-phase column (Nucleosil C18, 5 microns) with an acidic mobile phase (methanol-phosphate buffer, pH 2) containing an aliphatic tertiary amine. UV detection at 308 or 254 nm was used. The limit of quantitation was 5 ng/ml using 3.00 ml of plasma and detection at 254 nm. The intra-assay precision for bromhexine was better than 3.6% at 5 ng/ml.

Determination of cysteine, glutathione and N-acetylcysteine in plasma by ion-pair reversed-phase liquid chromatography and post-column derivatization.

The thiol and oxidized forms of cysteine, glutathione and N-acetylcysteine in plasma were determined by ion-pair liquid chromatography and post-column derivatization. The thiol forms were measured after direct injection of deproteinized plasma. The oxidized forms, present either as a dimer or oxidized with other small thiols, were assayed in deproteinized plasma after reduction with dithiothreitol. The total amounts, including the fraction bound to plasma proteins via disulphide bonds, were determined after reductive cleavage in plasma with dithiothreitol. The compounds were separated by ion-pair liquid chromatography on a reversed-phase column (C18) and were detected by fluorimetry after post-column derivatization. The endogenous plasma levels of all forms of cysteine, glutathione and N-acetylcysteine, except for the thiol form of N-acetylcysteine, were above the quantification limits. The quantification limit of N-acetylcysteine as a thiol, was 0.15 microM. The precision was better than 12% for the endogenous concentrations.

Effect of long-term administration of manganese on biogenic amine levels in discrete striatal regions of rat brain.

The effect of long-term manganese exposure of rats on biogenic amine levels in striatal brain regions is described. Four groups of male Sprague-Dawley rats received manganese as MnCl2 continuously in the drinking water for 60, 100, 165 and 265 days, respectively. Discrete regions within the caudate-putamen were punched out. Dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5-hydroxyindoleacetic acid were determined by high performance liquid chromatography with electrochemical detection. Rats exposed for 60 and 165 days showed significantly increased levels of dopamine and 3,4-dihydroxyphenylacetic acid in discrete regions of the dorsal caudate-putamen. The affected regions were possibly not identical in the two age groups but they were adjacently situated. These alterations were not found in rats exposed for 100 or 265 days.